The study of protein structure and function has historically relied upon the properties and reaction chemistries that are available using the reactive groups of the naturally occurring amino acids. Unfortunately, every known organism, from bacteria to humans, encodes the same twenty common amino acids. These 20 amino acids comprise a surprisingly limited number of functional groups: nitrogen bases, carboxylic acids and amides, alcohols, and a thiol group. This limited selection of R-groups has restricted the study of protein structure and function, where the studies are confined by the chemical properties of the naturally occurring amino acids. For example, the limited number of naturally occurring reactive R-groups has limited the ability to make highly targeted protein modifications to the exclusion of the other amino acids in a protein.
Chemoselective ligation reactions involving proteins are extremely important for a variety of purposes, including but not limited to studying protein-protein interaction and cellular signaling, and generating novel protein therapeutics. Most selective protein modification reactions currently used in the art involve covalent bond formation between nucleophilic and electrophilic reaction partners that target naturally occurring nucleophilic residues in the protein amino acid side chains, e.g., the reaction of α-halo ketones with histidine or cysteine side chains. Selectivity in these cases is determined by the number and accessibility of the nucleophilic residues in the protein. Unfortunately, naturally occurring proteins frequently contain poorly positioned (e.g., inaccessible) reaction sites or multiple reaction targets (e.g., lysine, histidine and cysteine residues), resulting in poor selectivity in the modification reactions, making highly targeted protein modification by nucleophilic/electrophilic reagents difficult. Furthermore, the sites of modification are typically limited to the naturally occurring nucleophilic side chains of lysine, histidine or cysteine. Modification at other sites is difficult or impossible.
What is needed in the art are new strategies for incorporation of unnatural amino acids into proteins for the purpose of modifying and studying protein structure and function, where the unnatural amino acids have novel reaction chemistries or other properties, e.g., biological properties not found in the naturally occurring amino acids. There is a considerable need in the art for the creation of new strategies for protein modification reactions that modify proteins in a highly selective fashion, and furthermore, modify proteins under physiological conditions. What is needed in the art are novel methods for producing protein modifications, where the modifications are highly specific, e.g., modifications where none of the naturally occurring amino acids are subject to cross reactions or side reactions. Novel chemistries for highly specific protein modification strategies find a wide variety of applications in the study of protein structure and function and in the production of therapeutic proteins.
Protein Lipidation
Protein lipidation is a key post-translational modification that is involved in protein localization, proper intracellular protein trafficking and protein-protein interactions. Lipidation of proteins is frequently required for proper biological activity. This feature is critical for the development of some therapeutic proteins. Lipidation is also critical in studying protein-protein interactions and cellular signaling. Unfortunately, in vitro chemoselective ligation to produce lipidated proteins using the native unlipidated form of the protein is extremely difficult, and is generally limited to modification of unique surface exposed cysteine residues.
The biological activities of many cellular proteins require association with the cell membrane, which is dependent on the post-translation modification of cysteine by lipid residues such as farnesyl, myristoyl, and palmitoyl moeties (Chernomordik and Kozlov (2003), Annual Review of Biochemistry 72:175-207). For example, many G-protein coupled receptors are palmitoylated, Ras proteins are both farnesylated and palmitoylated (Chernomordik and Kozlov (2003), Annual Review of Biochemistry 72:175-207). While protein farnesylation is a stable and irreversible modification, palmitoylation is reversible, resulting in dynamic regulation of protein function, and specific targeting to cellular membranes (Rocks et al. (2005), Science 307(5716): 1746-1752). Furthermore, γ-carboxyglutamic acid is an essential modification that is important for calcium-dependent membrane adhesion in the coagulation cascade (Davie et al. (1991), Biochemistry 30(43): 10363-10370).
Orthogonal Translation Systems
One strategy to overcome the limitations of a limited genetic code is to expand the genetic code and add amino acids that have novel reactive properties to the biological repertoire. A general methodology has been developed for the in vivo site-specific incorporation of diverse unnatural amino acids into proteins in both prokaryotic and eukaryotic organisms. These methods rely on orthogonal protein translation components that recognize a suitable selector codon to insert a desired unnatural amino acid at a defined position during polypeptide translation in vivo. These methods utilize an orthogonal tRNA (O-tRNA) that recognizes a selector codon, and where a corresponding specific orthogonal aminoacyl-tRNA synthetase (an O-RS) charges the O-tRNA with the unnatural amino acid. These components do not cross-react with any of the endogenous tRNAs, RSs, amino acids or codons in the host organism (i.e., it must be orthogonal). The use of such orthogonal tRNA-RS pairs has made it possible to genetically encode a large number of structurally diverse unnatural amino acids.
The practice of using orthogonal translation systems that are suitable for making proteins that comprise one or more unnatural amino acid is generally known in the art, as are the general methods for producing orthogonal translation systems. For example, see International Publication Numbers WO 2002/086075, entitled “METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS;” WO 2002/085923, entitled “IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS;” WO 2004/094593, entitled “EXPANDING THE EUKARYOTIC GENETIC CODE;” WO 2005/019415, filed Jul. 7, 2004; WO 2005/007870, filed Jul. 7, 2004; WO 2005/007624, filed Jul. 7, 2004 and WO 2006/110182, filed Oct. 27, 2005, entitled “ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVO INCORPORATION OF UNNATURAL AMINO ACIDS.” Each of these applications is incorporated herein by reference in its entirety. For additional discussion of orthogonal translation systems that incorporate unnatural amino acids, and methods for their production and use, see also, Wang and Schultz, “Expanding the Genetic Code,” Chem. Commun. (Camb.) 1:1-11 (2002); Wang and Schultz “Expanding the Genetic Code,” Angewandte Chemie Int. Ed., 44(1):34-66 (2005); Xie and Schultz, “An Expanding Genetic Code,” Methods 36(3):227-238 (2005); Xie and Schultz, “Adding Amino Acids to the Genetic Repertoire,” Curr. Opinion in Chemical Biology 9(6):548-554 (2005); Wang et al., “Expanding the Genetic Code,” Annu. Rev. Biophys. Biomol. Struct., 35:225-249 (2006); and Xie and Schultz, “A Chemical Toolkit for Proteins—an Expanded Genetic Code,” Nat. Rev. Mol. Cell. Biol., 7(10):775-782 (2006).
There is a need in the art for the development of orthogonal translation components that incorporate unnatural amino acids into proteins, where the unnatural amino acids can be incorporated at a defined position, and where the unnatural amino acids have novel chemical properties that allow the amino acid to serve as a target for specific modification (e.g., lipidation) to the exclusion of cross reactions or side reactions with other sites in the proteins. The invention described herein fulfills these and other needs, as will be apparent upon review of the following disclosure.